DIRECT ASSAY FOR O-6-METHYLGUANINE-DNA METHYLTRANSFERASE AND COMPARISON OF DETECTION METHODS FOR THE METHYLATED ENZYME IN POLYACRYLAMIDE GELS AND ELECTROBLOTS

We describe in detail a direct assay for the substrate-inactivated DNA-repair enzyme. O6-methylguanine-DNA methyltransferase (O6-MT), which measures the transfer of radiolabelled methyl groups from a prepared O6-methylguanine-DNA substrate to the protein fraction of an enzyme-containing cell/tissue extract. This assay, a modification of a previously suggested method for monitoring O6-ethylguanine-DNA repair [Renard, Verly, Mehta & Ludlum (1983) Eur. J. Biochem. 136, 461-467], is sensitive, highly reproducible, accurate and is, as described here and relative to previously published methods, well suited for use with a large number of test samples. We identified two problems with the O6-[Me-H-3]methylguanine-DNA substrate used in the present work and in other reported assays: firstly, that of progressively higher assay backgrounds with increasing age of substrate, which was nullified by once-only purification of the double-stranded substrate by hydroxyapatite chromatography; secondly, a substrate of high specific radioactivity (30 Ci/mmol), made with freshly prepared tritiated methylnitrosourea, behaved as a substrate of 5 Ci/mmol when referenced against radiolabelled O6-methylguanine-DNA made with either [H-3]- or [C-14]-methylnitrosourea at the lower specific radioactivities of 1 Ci/mmol and 61 mCi/mmol respectively. This apparently stemmed from the known instability of high-specific-radioactivity [H-3]methylnitrosourea and indicated that an expected increase in sensitivity of the assay does not necessarily result from increasing the specific radioactivity of substrates above approx. 1 Ci/mmol. Although O6-MT was stable to preincubation at 25-degrees-C, marked losses of activity were observed at 37-degrees-C, and more so at 45-degrees-C. Enzyme lability at the higher temperatures was not, however, seen during preincubation in the presence of its substrate, O6-[Me-H-3]methylguanine-DNA, which apparently protected O6-MT against thermal inactivation. As previously seen with other human cells and tissues, extracts of human spleen in the present study showed wide interindividual differences in O6-MT specific activity (18-fold), which spanned the range 50-900 fmol/mg of protein. Cultured human lymphoblastoid Jurkat cells contained approx. 57000 enzyme molecules/cell. Substrate-inactivated [Me-H-3]methylated O6-MT was analysed by SDS/PAGE and electroblotting. The different but similarly sized forms of this enzyme that we previously detected in human spleen [Major, Gardner, Carne & Lawley (1990) Nucleic Acids Res. 18, 1351-1359] were clearly resolved by fluorography of electroblots, but only at considerable expense of time. As expected, scintillation counting of the protein extracted from gel slices and linear-wire scanning of enzyme-associated radioactivity on electroblots were higher methods for detecting the [Me-H-3]methylated inactivated O6-MT.